Water quality monitoring based on chemometric analysis of high-resolution phytoplankton data measured with flow cytometry.

River water is an important source of Dutch drinking water. For this reason, continuous monitoring of river water quality is needed. However, comprehensive chemical analyses with high-resolution gas chromatography [GC]-mass spectrometry [MS]/liquid chromatography [LC]-MS are quite tedious and time consuming; this makes them poorly fit for routine water quality monitoring and, therefore, many pollution events are missed. Phytoplankton are highly sensitive and responsive to toxicity, which makes them highly usable for effect-based water quality monitoring. Flow cytometry can measure the optical properties of phytoplankton every hour, generating a large amount of information-rich data in one year. However, this requires chemometrics, as the resulting fingerprints need to be processed into information about abnormal phytoplankton behaviour. We developed Discriminant Analysis of Multi-Aspect CYtometry (DAMACY) to model the "normal condition" of the phytoplankton community imposed by diurnal, meteorological, and other exogenous influences. DAMACY first describes the cellular variability and distribution of phytoplankton in each measurement using principal component analysis, and then aims to find subtle differences in these phytoplankton distributions that predict normal environmental conditions. Deviations from these normal environmental conditions indicated abnormal phytoplankton behaviour that happened alongside pollution events measured with the GC/MS and LC/MS systems. Thus, our results demonstrate that flow cytometry in combination with chemometrics may be used for an automated hourly assessment of river water quality and as a near real-time early warning for detecting harmful known or unknown contaminants. Finally, both the flow cytometer and the DAMACY algorithm run completely autonomous and only requires maintenance once or twice per year. The warning system results may be uploaded automatically, so that drinking water companies may temporary stop pumping water whenever abnormal phytoplankton behaviour is detected. In the case of prolonged abnormal phytoplankton behaviour, comprehensive analysis may still be used to identify the chemical compound, its origin, and toxicity.

Optimizing the setup of a flow cytometric cell sorter for efficient quantitative sorting of long filamentous cyanobacteria.

Heterogeneity within natural phytoplankton communities makes it very difficult to analyze parameters at the single-cell level. Flow cytometric sorting is therefore a useful tool in aquatic sciences, as it provides material for post-sort analysis and culturing. Sorting subpopulations from natural communities, however, often requires handling morphologically diverse and complex particles with various abundances. Long particles, such as filament-forming cyanobacteria (>100-µm long), prove very difficult to handle. These potentially toxic organisms are widespread in eutrophic systems and have important ecological consequences. Being able to sort filamentous cyanobacteria efficiently and as viable cells is therefore highly desirable when studying factors associated with their toxicity and occurrence. This unconventional sorting requires extensive user experience and special instrument setup. We have investigated the effect of hydrodynamic and electromechanical components of a flow cytometer, and sorting protocol on the quantitative sorting efficiency of these long particles using two filamentous cyanobacterial strains with average lengths of ∼100 and ∼300 µm. Sorting efficiency ranged from 9.4 to 96.0% and was significantly affected by filament length, sorting envelope, drop delay (dd), and for the long species also by tip size, but not by cycle time. Filaments survived sorting and were not damaged. The optimal settings found for the modular MoFlo® cell-sorter to sort the filaments were a 100-µm flow tip at 30 psi (207 kPa) with a three-droplet envelope in Enrich mode while using an extended analysis time of 17.6 µs and an intermediate plate charge and deflection percentage combination of 3,000 V/60%, combined with a dd 0 for the cultures with 100-µm filaments and dd +1 for the culture with 300-µm filaments. To the best of our knowledge, the filaments up to 1063.5 µm sorted in this study are the longest ever sorted.

Adaptive divergence in pigment composition promotes phytoplankton biodiversity.

The dazzling diversity of the phytoplankton has puzzled biologists for decades. The puzzle has been enlarged rather than solved by the progressive discovery of new phototrophic microorganisms in the oceans, including picocyanobacteria, pico-eukaryotes, and bacteriochlorophyll-based and rhodopsin-based phototrophic bacteria. Physiological and genomic studies suggest that natural selection promotes niche differentiation among these phototrophic microorganisms, particularly with respect to their photosynthetic characteristics. We have analysed competition for light between two closely related picocyanobacteria of the Synechococcus group that we isolated from the Baltic Sea. One of these two has a red colour because it contains the pigment phycoerythrin, whereas the other is blue-green because it contains high contents of the pigment phycocyanin. Here we report theory and competition experiments that reveal stable coexistence of the two picocyanobacteria, owing to partitioning of the light spectrum. Further competition experiments with a third marine cyanobacterium, capable of adapting its pigment composition, show that this species persists by investing in the pigment that absorbs the colour not used by its competitors. These results demonstrate the adaptive significance of divergence in pigment composition of phototrophic microorganisms, which allows an efficient utilization of light energy and favours species coexistence.